Simple Bradford Assay Question - water dilutions - (Mar/03/2008 )

Hi everyone,

I just had a quick question about the Bradford assay. I use the microtitre method (96 well plate).
I just wanted to know if it's ok to dilute your standards and extracted protein in water, or if you should use buffer? I've been doing it with water and it's generally worked pretty well.....however I have some really important samples (extracting protein from WBC) and equal loading will make or break the experiment,.

I know some people use McIlvaines buffer to dilute their protein into for a bradford assay.

what do you think? who uses water, who uses buffer? what buffer and why?

thanks in advance

Our lab uses 0.15M NaCl

hope this helps!

we use water but we also run buffer blanks (with sample buffer treated the same way as the sample) and the readings subtracted from the samples.

We use water for standards, that way we can use the same standards for different samples. In addition we run a buffer blank (buffer only) to check for any interference with the assay, and subtract any abs of this from the abs of the samples.

Good luck!

Whatever you do, you keep the same for samples and standards with reagard to buffer/detergent compositions, as these are critical to the readings.

Thanks everyone!

Especially to whoever answered first.

I ran a test today and prepared sample standards using water and using the 0.15 NaCl

my R2 value for the BSA sample diluted in water (average triplicates) was 0.91
my R2 value for the BSA samples diluted in NaCl (average triplicates) was 0.99!!!

Do you know why this is?? does the NaCl help with protein folding so the biorad recognizes the protein better?

I also used my lysis buffer as a control and got ~ the same absorbance as water so i don't think there is anything interfering.....so I think I'm ready to go